Piroplasmosis in Wildlife: Babesia and Theileria Affecting Free-Ranging

Zanet et al. Parasites & Vectors 2014, 7:70 http://www.parasitesandvectors.com/content/7/1/70

RESEARCH Open Access Piroplasmosis in wildlife: Babesia and Theileria affecting free-ranging ungulates and carnivores in the Italian Alps Stefania Zanet1, Anna Trisciuoglio1, Elisa Bottero1, Isabel Garcia Fernández de Mera2, Christian Gortazar2, Maria Grazia Carpignano3 and Ezio Ferroglio1*

Abstract Background: Piroplasmosis are among the most relevant diseases of domestic animals. Babesia is emerging as cause of tick-borne zoonosis worldwide and free-living animals are reservoir hosts of several zoonotic Babesia species. We investigated the epidemiology of Babesia spp. and Theileria spp. in wild ungulates and carnivores from Northern Italy to determine which of these apicomplexan species circulate in wildlife and their prevalence of infection. Methods: PCR amplification of the V4 hyper-variable region of the 18S rDNA of Babesia sp./Theileria sp was carried out on spleen samples of 1036 wild animals: Roe deer Capreolus capreolus (n = 462), Red deer Cervus elaphus (n = 52), Alpine Chamois Rupicapra rupicapra (n = 36), Fallow deer Dama dama (n = 17), Wild boar Sus scrofa (n = 257), Red fox Vulpes vulpes (n = 205) and Wolf Canis lupus (n = 7). Selected positive samples were sequenced to determine the species of amplified Babesia/Theileria DNA. Results: Babesia/Theileria DNA was found with a mean prevalence of 9.94% (IC95% 8.27-11.91). The only piroplasms found in carnivores was Theileria annae, which was detected in two foxes (0.98%; IC95% 0.27-3.49). Red deer showed the highest prevalence of infection (44.23%; IC95% 31.6-57.66), followed by Alpine chamois (22.22%; IC95% 11.71-38.08), Roe deer (12.55%; IC95% 9.84-15.89), and Wild boar (4.67%; IC95% 2.69-7.98). Genetic analysis identified Babesia capreoli as the most prevalent piroplasmid found in Alpine chamois, Roe deer and Red deer, followed by Babesia bigemina (found in Roe deer, Red deer and Wild boar), and the zoonotic Babesia venatorum (formerly Babesia sp. EU1) isolated from 2 Roe deer. Piroplasmids of the genus Theileria were identified in Wild boar and Red deer. Conclusions: The present study offers novel insights into the role of wildlife in Babesia/Theileria epidemiology, as well as relevant information on genetic variability of piroplasmids infecting wild ungulates and carnivores. Keywords: Piroplasmosis, Babesia, Theileria, PCR, Wildlife, Italy

Background Theileria spp. are considered to be benign or less patho- Babesia spp. and Theileria spp. are protozoan parasites genic probably because of a long evolutionary relationship transmitted mainly by ticks and able to infect erythrocytes between the parasite and the host. Nevertheless, clinical and/or leukocytes of a wide variety of domestic and wild disease may occur in stressful situations related to trans- animals [1] and Babesia is the second most common para- location of wildlife or/and when the host is debilitated by site found in the blood of mammals after trypanosomes [2]. other parasitic organisms or malnutrition [5-7]. Although Some species of the genus Theileria such as T. annulata piroplasmosis is among the most relevant diseases of do- and T. parva are highly pathogenic to cattle and cause sig- mestic animals [8,9], many unknowns remain concerning nificant mortality among susceptible animals [3,4]. Other their epidemiology and life cycle in the Ixodid tick vector as well as in the vertebrate host, especially for factors involved in the role of wildlife as reservoirs of infection * Correspondence: [email protected] 1Department of Veterinary Sciences, University of Turin, via Leonardo da [2,10]. Recently, white-tailed deer were indicated as being Vinci, 44, 10095 Grugliasco, TO, Italy responsible for reintroducing the tick vector of Cattle Tick Full list of author information is available at the end of the article

© 2014 Zanet et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zanet et al. Parasites & Vectors 2014, 7:70 Page 2 of 7 http://www.parasitesandvectors.com/content/7/1/70

Fever in Central Texas and hence the piroplasms respon- identifications of this parasite attributing many of them to sible for bovine babesiosis (Babesia bovis and Babesia B. capreoli. In contrast to ungulates, information on the bigemina) [11]. Different Theileria and Babesia species occurrence and prevalence of piroplasmids in wild canids were reported in wildlife with high prevalence of infection is limited [34]. In Europe, a continent where the Red fox [5,9,12], some of which are recognized zoonotic patho- is present at high densities [35], B. microti-like piroplasms gens, such as Babesia divergens [13], Babesia divergens- were molecularly confirmed in foxes from Central and like [14,15], Babesia venatorum (formerly Babesia sp. Northern Spain [9,36,37], Croatia [38] and Portugal [34]. EU1) [16-18], Babesia microti [2,19,20] and B. microti-like In Portugal, a single fox was also found infected with [21,22]. No Theileria species have to date been identified B. canis [34]. Considering the relevant insights into as causative agents of zoonotic infections [2]. The epi- piroplasm-wildlife epidemiology [39,40], the increasing demiology of Babesia and Theileria in European wildlife is importance of Babesia as an emerging zoonotic disease complex. Besides the variety of host species susceptible to [2,10] and the lack of information on piroplasm epide- infection (i.e. cervids: Roe deer Capreolus capreolus,Red miology in Northwestern Italy [23], we widely investigated deer Cervus elaphus, Fallow deer Dama dama; bovids: Babesia/Theileria infection prevalence in wild ungulates Alpine Chamois Rupicapra rupicapra, Spanish Ibex Capra and carnivores from the Piedmont region (Italy) and their pyrenaica;suids:wildboarSus scrofa) [5,23-25], different biomolecular characteristics, by amplifying and sequen- tick vectors contribute to piroplasmid transmission: Ixodes cing part of the V4 hyper-variable region of the 18S rRNA ricinus is the most common vector of Babesia species in gene. Europe but recently, 5 species of ticks were found to be infected with piroplasmids in Central and Northern Italy Methods [26], suggesting the need to investigate other potential A total of 1036 free-ranging wild ungulates and carnivores vector species since novel tick/pathogen associations were were sampled during a period of 5 years (from 2008 to detected in Hyalomma marginatum, Rhipicephalus san- 2012), in a wide area of Northern Italy (2 million hectares), guineus and I. ricinus [26]. In North America, I. scapularis that includes a wide range of habitats and different eco- is the primary vector responsible for transmission of logical niches ranging from low altitude intensively cul- B. microti to humans [2]. As for Babesia,theoccurrence tivated farm land to Alpine forests and meadows. The of Theileria depends on the simultaneous presence of animals included in the study were either culled by appropriate vector ticks and host species. In the USA, the hunters, accidentally found dead, or culled within official lone-star tick Ambloymma americanum is the only known programs for species demographic control. Ethical and vector of T. cervi, which mainly infects wild ruminants, institutional approval was given by the Department of particularly White-tailed deer [27,28]. In Europe, little is Veterinary Sciences, University of Turin (Italy). Six species known about tick species involved in the transmission of of ungulates were sampled: Roe deer Capreolus capreolus Theileria to cervids, and it is assumed that ticks of the (n = 462), Red deer Cervus elaphus (n = 52), Alpine cha- genera Ixodes, Hyalomma and Rhiphicephalus might be mois Rupicapra rupicapra (n = 36), Fallow deer Dama involved [5,7]. As biomolecular tools are easily available, dama (n = 17) and Wild boar Sus scrofa (n = 257). Two in recent years many studies have investigated the epizoo- carnivore species, Red fox Vulpes vulpes (n = 205) and tiology of piroplasmids circulating in wildlife, leading to Wolf Canis lupus (n = 7) were also included in the study. the identification of several Babesia and Theileria species All animals were promptly brought to the Turin Veteri- strictly related to wild animals or of strains shared with nary Faculty for necropsy where a portion of splenic tissue domestic animals [11,29]. The novel B. venatorum is was collected from each animal and stored at −20°C until strictly related to Roe deer presence [24], while B. capreoli further processing. All standard precautions were taken to was detected in Red deer from Ireland, where Roe deer minimize the risk of cross-contamination (preparation of are absent [24]. C. capreolus was also identified as a pos- PCR and addition of DNA was carried out in separate sible source of fatal babesial infection for the Alpine laminar-flow cabinets using DNA-free disposable material. chamois [30,31]. The territorial expansion and population Positive and negative control samples were processed in increase that involved Roe deer in several European coun- parallel with all samples). Total genomic DNA was ex- tries including Italy [32], led to increased contacts and tracted from ≈ 10 mg of spleen using the commercial kit spatial overlap with R. rupicapra populations and hence to GenElute® Mammalian Genomic DNA Miniprep (Sigma– an increased possibility of infection of this species with Aldrich, St. Louis, MO, USA) according to manufacturer’s piroplasms of cervids such as B. capreoli [23]. The zoo- instructions. Direct molecular detection of Babesia spp./ notic B. divergens is also reported to infect European Theileria spp. DNA was carried out using a semi-nested wild ungulates and has probably one of the largest host PCR protocol targeting the V4 hyper-variable region of ranges described to date for a Babesia species [33], al- the 18S ribosomal RNA gene. The primers used are highly though Malandrin et al. [13] recently reviewed previous conserved and can amplify a wide variety of Babesia and Zanet et al. Parasites & Vectors 2014, 7:70 Page 3 of 7 http://www.parasitesandvectors.com/content/7/1/70

Theileria species [41,42]. The first amplification was Table 1 Prevalence of Babesia/Theileria spp. in wildlife carried out using the primers RLB-F2 (5’-GACACAGG species GAGGTAGTGACAAG-3’) and RLB-R2 (5’-CTAAGAA Species Prevalence (IC95%) Positive/total sampled TTTCACCTCTGACAGT-3’) [41]. The PCR reaction was Roe deer 12.55% [9.84-15.89] 58/462 μ carried out in a final volume of 25 l, using Promega PCR Wild boar 4.67% [2.69/7.98] 12/257 Master Mix (Promega Corporation, WI, USA), 20 pM of Fallow deer 0.00% [0.00/18.43] 0/17 each primer, and ≈ 100 ng of DNA template. The amplification included a 5 min denaturation step at 95°C Alpine chamois 22.22% [11.71/38.08] 8/36 followed by 25 repeats of 30 s at 95°C, 45 s at 50°C, and Red deer 44.23% [31.6/57.66] 23/52 1.5 min at 72°C and a final extension at 72°C for 10 min. Red fox 0.98% [0.27/3.49] 2/205 Amplicons (1 μl) of the first PCR step were used as tem- Grey Wolf 0.00% [0.00/35.43] 0/7 plate for the second amplification which used RLB-FINT Total 9.94% [103/1036] 103/1036 ’ ’ (5 -GACAAGAAATAACAATACRGGGC-3 )[43]asin- PCR prevalence to Babesia/Theileria differs greatly among sampled species. We ternal forward primer, together with RLB-R2. The reaction reported detailed prevalence values and confidence intervals (95%), together mixture and cycling program were identical to the direct with the number of tested and positive animals of each sampled species. RLB-F2/RLB-R2 amplification, but the cycle number was increased to 40, whereas the annealing temperature was shown), nor any statistically significant difference between 50°C in the first PCR and 55°C in the second PCR. A posi- sampling years was recorded (data not shown). tive and a negative control sample were included in each amplification reaction. Amplicons were analyzed by aga- Sequencing and molecular classification rose gel electrophoresis (2%) and visualized by staining A total of 28 positive samples were sequenced and depos- with Gel Red Nucleic Acid Gel Stain (VWR International ited in GenBank under accession numbers from KF773715 Milano, Italy). Selected positive amplicons were purified to KF773741 (Table 2). B. capreoli was the most prevalent (QIAquick PCR purification kit, QIAGEN) and both DNA species (P = 46.43%; CI95% 29.53-64.19), it was found in strands were directly sequenced (Macrogen; http://www. Alpine chamois (n = 4), Roe deer (n = 4), and Red deer macrogen.com). The resulting sequences were compared (n = 5). All four B. capreoli isolates from Alpine chamois with the NCBI/Genbank database using the Basic Local [GenBank: from KF773728 to KF773730] showed 100% Alignment Search Tool (BLAST), and ClustalX software identity with the B. capreoli described by Hoby et al., [30] (http://www-igbmc.u-strasbg.fr/BioInfo) was used to con- in fatal cases of babesiosis in chamois from Switzerland struct multiple-sequence alignments. All statistical analysis [GenBank:EF545558 to EF545562], while a Red deer iso- was carried out using R software 3.0.1 (R Core Team late [Genbank: KF773718] showed 100% homology with 2012). Chi-square test was used to assess potential diffe- B. capreoli [GenBank: FJ944827- FJ944828] identified in rences in Babesia/Theileria infection rate between groups Roe deer in France [13]. B. bigemina was the second most (animals were grouped by species, age, sex, and year of prevalent Babesia species. It was identified in Roe deer sampling). (n = 4), Red deer (n = 1) and Wild boar (n = 2). All isolates showed 99% identity with B. bigemina [Genbank: Results HQ264116], infecting White-tailed deer in Southern Texas Prevalence of Babesia/Theileria spp. DNA in wildlife [11]. Roe deer (n = 2) were also infected with the zoonotic Piroplasm DNA was detected with an overall prevalence of B. venatorum. Both isolates showed 100% identity with the 9.94% (IC95% 8.27-11.91). Detailed prevalence data for each B. venatorum [GenBank: AY046575] described in two species are reported in Table 1. Herbivores (P = 15.7%; human cases in Italy and Austria [16]. Piroplasms of the IC95% 12.93-18.92) were significantly more infected (χ2 = genus Theileria were detected in Wild boar (n = 2) and 32.55, p < 0.0000; OR = 19.55, 4.77-80.14) than carnivores Red deer (n = 2). Wild boar isolates showed 100% identity (P = 0.94%; IC95% 0.26-3.37) as Babesia/Theileria DNA with Theileria sp. CS-2012 [Genbank: JQ751279] des- was amplified from two foxes, while all the results from the cribed in Wild boars and Sambar deer in Thailand. An iso- wolves examined were negative by PCR. Among herbivores, late from Red deer [Genbank: KF773724] showed 100% the highest prevalence of infection was reported in Red identity with Theileria sp. 3185/02 described in a Red deer deer (P = 44.23%; IC95% 31.6-57.66). This species was sig- imported from Germany to Spain [Genbank: AY421708] nificantly more infected than the other examined ungulates [7], while another Red deer isolate [GenBank: KF773725] (χ2 = 51.06, p < 0.0000; OR = 6.88, 3.8-12.48). On the other showed 100% identity with Theileria sp. OT3 described hand, Wild boar results showed significantly less infection in chamois, deer and sheep from Spain [GenBank: than herbivore species (χ2 = 20.00, p < 0.0000; OR = 0.2631, DQ866840] [44,45]. T. annae (syn. B. microti-like) was 0.14-0.49). Sex and age did not significantly influence the detected in the two PCR positive foxes. One isolate infection status of any of the species tested (data not [GenBank: KF773740] showed 100% identity with T. annae Zanet et al. Parasites & Vectors 2014, 7:70 Page 4 of 7 http://www.parasitesandvectors.com/content/7/1/70

Table 2 Babesia/Theileria species identification Host Area of origin Babesia/Theileria specie GenBank accession no. Chamois [R. rupicapra] SW Piedmont, Cuneo Province B. capreoli KF773728* SW Piedmont, Cuneo Province B. capreoli KF773729 SW Piedmont, Cuneo Province B. capreoli KF773730 NW Piedmont, Torino Province B. capreoli KF773728* Red deer [C. elaphus] NW Piedmont, Torino Province B. capreoli KF773718 NW Piedmont, Torino Province Theileria sp. KF773724 SE Piedmont, Alessandria Province Theileria sp. KF773725 NW Piedmont, Torino Province B. bigemina KF773727 NW Piedmont, Torino Province B. capreoli KF773733 SW Piedmont, Cuneo Province B. capreoli KF773734 NE Piedmont, Biella Province B. capreoli KF773735 SE Piedmont, Alessandria Province B. capreoli KF773736 Roe deer [C. capreolus] SE Piedmont, Alessandria Province B. bigemina KF773715 NW Piedmont, Torino Province B. bigemina KF773719 SW Piedmont, Cuneo Province B. bigemina KF773720 SW Piedmont, Cuneo Province B. bigemina KF773721 NW Piedmont, Torino Province B. venatorum KF773722 NW Piedmont, Torino Province B. capreoli KF773723 SE Piedmont, Alessandria Province B. capreoli KF773724 SE Piedmont, Alessandria Province B. capreoli KF773731 NW Piedmont, Torino Province B. venatorum KF773732 SW Piedmont, Cuneo Province B. capreoli KF773737 Wild boar [S. scrofa] SE Piedmont, Alessandria Province B. bigemina KF773716 NW Piedmont, Torino Province B. bigemina KF773717 SE Piedmont, Alessandria Province Theileria sp. KF773738 SE Piedmont, Alessandria Province Theileria sp. KF773739 Red fox [V. vulpes] NW Piedmont, Torino Province T. annae KF773740 NW Piedmont, Torino Province T. annae KF773741 Babesia/Theileria species was determined, for selected positive samples, by sequencing of PCR products. For each of the five animal species in which piroplasmid DNA was detected we reported the parasite species, the GenBank accession number attributed to each isolate and the area of origin. *A unique accession number was attributed to two chamois. amplified from Croatian foxes [GenBank: HM212628] Babesia and Theileria are traditionally known for their [38], while the second isolate [Genbank: KF773741], al- relevant economic impact on the livestock industry and though very similar, has CT instead of AA at 268–269 pb. on human health. Several wild animals are known to be The phylogenetic relationships among the sequenced sam- reservoirs of zoonotic Babesia species [2]. Although piro- ples are reported in Figure 1. plasmosis in wildlife is mostly asymptomatic [46] recent cases of fatal babesiosis were recorded in Alpine chamois Discussion infected with B. capreoli [30,31]. Malandrin et al., [13] Piroplasmids are frequently found to infect free-living clarified the ambiguous classification of B. divergens-like animals worldwide and are gaining increasing attention parasites isolated from cervids, revealing that most in- as emerging tick-borne zoonosis [16,46,47]. Increased fections reported from wildlife can likely be ascribed to wildlife-human interactions due to socio-economic chan- B. capreoli. Amplification and sequencing of the 18S ges have enhanced the risk of contracting zoonotic rRNA gene was essential to correctly identify the two spe- diseases [23]. This is especially true for vector-borne cies (B. divergens and B. capreoli) as distinct. PCR proto- pathogens where the presence of the vector in the area cols targeting the V4 hyper-variable region of 18S rDNA, has increased thanks to climatic changes and/or changes like the one used in the present study, are able to identify in the environment or in host species presence [48-50]. awidevarietyofBabesia/Theileria species and may also Zanet et al. Parasites & Vectors 2014, 7:70 Page 5 of 7 http://www.parasitesandvectors.com/content/7/1/70

Figure 1 Phylogenetic tree of the Babesia/Theileria isolates from carnivores and ruminants of Northwestern Italy performed using Neighbor-joining and 1000 bootstrap replicates. The samples were coded with their corresponding GenBank accession number. detect less closely related genotypes, such as the B. microti of a common epidemiological cycle among wildlife and complex or species related to B. odocoilei [29]. In our sympatric livestock, since cattle are the recognized res- study no B. divergens was identified in any of the se- ervoir of this parasite [11,53]. The prevalence of infec- quenced samples, while the closely related B. capreoli was tion recorded in our study differs greatly between the the most prevalent species, confirming that where Roe species considered. Red deer showed the highest preva- deer are abundant, B. capreoli is also widely present. lence of infection with 44% of sampled Red deer positive B. venatorum was first identified in splenectomized hu- by PCR. Prevalences reported for the same species in man patients in Italy and Austria [16] and in Roe deer Italy (12.5%) [23] as well as in other countries are sig- from Eastern Italy [23,51], but to our knowledge this is nificantly lower (in Ireland 26%) [24], in USA 12% [11]. the first report of B. venatorum from the Western Alps. Many Red deer populations present in Piedmont are The high homology found between the 18S rDNA se- characterized by high densities of animals [54]. In par- quences of B. venatorum deposited in Genbank suggests ticular, 62.5% of Babesia-infected Red deer came from a that the parasite widely circulates among wild ungulates low altitude fenced forest (Parco Regionale La Mandria; across Europe. Over the past 30 years, reintroduction of 3600 ha) mainly composed of broad-leaved trees and both Red deer and Roe deer occurred for restocking pur- permanent meadows where the estimated Red deer poses from Central Europe and from the Balkans to the population is very high (6–14 heads/km2) [55]. Tick- studied area [52] and the founding effect of animal trans- favorable conditions together with the high density location could also be implied for the high homology of population and gregarious habits of C. elaphus are pos- B. venatorum found across the Alps. In consideration of sible causes of the high infection rate we found in Red B. bigemina, in the current study, it was detected in Roe deer. The Roe deer, even if less gregarious and territorial deer, Red deer and Wild boar. Even if B. bovis and B. [56], is the second most infected species in Piedmont, bigemina infection have been already reported in confirming the high susceptibility of C. capreolus to piro- White-tailed deer from North America [11], to our plasmid infection. The prevalence of Babesia/Theileria knowledge this is the first report of B. bigemina infec- DNA we found in Piedmont (12.55%) falls within the ting wild ungulates in Europe, suggesting the existence range reported for the species in Italy and in other Zanet et al. Parasites & Vectors 2014, 7:70 Page 6 of 7 http://www.parasitesandvectors.com/content/7/1/70

European countries [17,23,29]. Our data also confirmed References the low susceptibility of wild boar which were found in- 1. Duh D, Punda-Polić V, Trilar T, Avšič-Županc T: Molecular detection of Theileria sp. in ticks and naturally infected sheep. Vet Parasitol 2008, fected at low prevalence (P = 4.67%), as reported also 151(2–4):327–331. from Eastern Italy (2.6%) [23]. T. annae is the only piro- 2. Yabsley MJ, Shock BC: Natural history of Zoonotic Babesia: role of wildlife plasmid we detected in Red foxes. Natural infection of reservoirs. Int J Parasitol 2013, 2:18–31. 3. Tait A, Hall FR: Theileria annulata: control measures, diagnosis and the V. vulpes with T. annae had already been documented potential use of subunit vaccines. Rev Sci Tech 1990, 9(2):387–403. withtheprevalenceofinfectionbeinghigherthanre- 4. Gitau GK, Perry BD, McDermott JJ: The incidence, calf morbidity and corded in the Western Alps, in several European coun- mortality due to Theileria parva infections in smallholder dairy farms in Murang’a District, Kenya. Prev Vet Med 1999, 39(1):65–79. tries including Italy (50% [1/2]) [23], Northern Spain 5. Sawczuk M, Maciejewska A, Skotarczak B: Identification and molecular (20% [1/5]) [37], Portugal (69.2% [63/91] [34] and characterization of Theileria sp. infecting red deer (Cervus elaphus)in Croatia (5.2% [10/191]) [38]. northwestern Poland. Eur J Wildl Res 2008, 54:225–230. 6. Kocan AA, Kocan KM: Tick-transmitted protozoan diseases of wildlife in North America. Bull Soc Vector Ecol 1991, 16:94–108. Conclusions 7. Höfle U, Vicente J, Nagore D, Hurtado A, Pena A, de la Fuente J, Gortazar C: The role of wildlife as reservoirs of zoonotic Babesia spe- The risks of translocating wildlife. Pathogenic infection with Theileria sp. cies, as well as the involvement of free-ranging ungulates and elaeophora elaphi in an imported red deer. Vet Parasitol 2004, 126:387–395. in the epidemiology of piroplasmids of veterinary impor- 8. Schnittger L, Yin H, Gubbels MJ, Beyen D, Niemann S, Jorgejan F, Ahmed JS: tance is well established [2,39,57]. Nevertheless, many Phylogeny of sheep and goat Theileria and Babesia parasites. Parasitol Res – gaps in host-piroplasmid and host-tick interactions re- 2003, 91:938 406. 9. Criado-Fornelio A, Martinez-Marcos A, Buling-Sarana A, Barba-Carretero JC: main. For many of the numerous Babesia/Theileria species, Molecular studies on Babesia, Theileria and Hepatozoon in southern some relevant biological aspects (e.g. reservoir hosts(s), and Europe. Part I. Epizootiological aspects. Vet Parasitol 2003, vectors(s)) as well as molecular characteristics remain 113:189–201. 10. Schnittger L, Rodriguez AE, Florin-Christensen M, Morrison DA: Babesia: poorly investigated. Yabsley et al., [2] reported how PCR- a world emerging. Inf Gen Evol 2012, 12:1788–1809. based studies with sequence analysis of piroplasms circu- 11. Holman PJ, Carroll JE, Pugh R, Davis DS: Molecular detection of Babesia lating in wildlife are needed to identify the causative bovis and Babesia bigemina in white-tailed deer (Odocoileus virginianuns) from Tom Green County in Central Texas. Vet Par 2011, 117:298–304. agents of novel cases of human babesiosis as well as for 12. Galuppi R, Aureli S, Bonoli C, Caffara M, Tampieri MP: Detection and managing effective surveillance plans on potential reser- molecular characterization of Theileria sp. in fallow deer (Dama dama) voirs and vectors. The data presented in this study give and ticks from an Italian natural preserve. Res Vet Sci 2011, 91:110–115. 13. Malandrin L, Jouglin M, Sun Y, Brisseau N, Chauvin A: Redescription of Babesia valuable insights on the piroplasms circulating in free- capreoli (Enigk and Friedhoff, 1962) from roe deer (Capreolus capreolus): ranging ungulates and carnivores in an extensive area isolation, cultivation, host specificity, molecular characterization and where wildlife lives in sympatry with a high-density hu- differentiation from Babesia divergens. Int J Parasitol 2010, 40:277–284. 14. Olmeda AS, Armstrong PM, Rosenthal BM, Valladares B, Del Castillo A, man and livestock population. De Armas F, Miguelez M, González A, Rodríguez Rodríguez JA, Spielman A, Telford SR III: A subtropical case of human babesiosis. Acta Trop 1997, Competing interests 67:229–234. The authors declare that they have no competing interests. 15. Qi C, Zhou D, Liu J, Cheng Z, Zhang L, Wang L, Wang Z, Yang D, Wang S, Chai T: Detection of Babesia divergens using molecular methods in Authors’ contributions anemic patients in Shandong Province, China. Parasitol Res 2011, SZ carried out the molecular studies with EB, participated in the sequence 109:241–245. analysis and wrote the first draft of the manuscript; AT participated and 16. Herwaldt BL, Cacciò S, Gherlinzoni F, Aspöck H, Slemenda SB, Piccaluga P, supervised the laboratory work, MGC directly provided many of the tested Martinelli G, Edelhofer R, Hollenstein U, Poletti G, Pampiglione S, chamois samples and organized sample collection in the field, IGFDM and Löschenberger K, Tura S, Pieniazek NJ: Molecular characterization of a CG collaborated to PCR setup and sample sequencing, EF coordinated the non-Babesia divergens organism causing zoonotic babesiosis in Europe. investigation, and finalized the manuscript. All authors read and approved Emerg Infect Dis 2003, 9(8):942–948. the final manuscript. 17. Duh D, Petrovec M, Bidovec A, Avis-Zupanc T: Cervids as Babesiae hosts, Slovenia. Emerg Infect Dis 2005, 11(7):1121–1123. Acknowledgments 18. Bonnet S, Jouglin M, L’Hostis M, Chauvin A: Babesia sp. EU1 from roe deer This study was partially supported by funds from the Piedmont Regional and transmission within Ixodes ricinus. Emerg Infect Dis 2007, Administration (“Convenzione tra la Regione Piemonte e la Facoltà di 13(8):1208–1210. Medicina Veterinaria dell’Università degli Studi di Torino per la 19. Anderson JF, Mintz ED, Gadbaw JJ, Magnarelli LA: Babesia microti, human razionalizzazione ed integrazione delle attività di raccolta e smaltimento babesiosis, and Borrelia bugdorferi in Connecticut. J Clin Microbiol 1991, degli animali selvatici morti o oggetto di interventi di contenimento”). The 29:2779–2783. authors would like to acknowledge the precious help of Drs. Davide Grande, 20. Telford SR 3rd, Spielman A: Reservoir competence of white-footed mice Angelo Romano and Giulio Falzoni for the support given to the project. for Babesia microti. J Med Entomol 1993, 30:223–227. 21. Matsui T, Inoue R, Kajimoto K, Tamekane A, Okamura A, Katayama Y, Author details Shimoyama M, Chihara K, Saito-ito A, Tsuji M: First documentation of 1Department of Veterinary Sciences, University of Turin, via Leonardo da transfusion-associated babesiosis in Japan. Rinsho Ketsueki 2000, Vinci, 44, 10095 Grugliasco, TO, Italy. 2SaBio IREC CSIC UCLM JCCM, Ronda de 41:628–634. Toledo s/n. 13071, Ciudad Real, Spain. 3Comprensorio Alpino Valli Grana e 22. Wei Q, Tsuji M, Zamoto A, Kohsaki M, Matsui T, Shiota T, Telford SR III, Maira, via Roma 28, 12025 Dronero, CN, Italy. Ishiara C: Human babesiosis in Japan: isolation of Babesia microti-like parasites from an asymptomatic transfusion donor and from a rodent Received: 13 December 2013 Accepted: 29 January 2014 from an area where babesiosis is endemic. J Clin Microbiol 2001, Published: 17 February 2014 39:2178–2183. Zanet et al. Parasites & Vectors 2014, 7:70 Page 7 of 7 http://www.parasitesandvectors.com/content/7/1/70

23. Tampieri MP, Galuppi R, Bonoli C, Cancrini G, Moretti A, Pietrobelli M: Wild species in a sheep population from Northern Spain. Int J Parasitol 2004, ungulates as Babesia hosts in northern and central Italy. Vector-Borne 34(9):1059–1067. Zoonot 2008, 8(5):667–674. 46. Homer MJ, Aguilar-Delfin I, Telford SRI, Krause PJ, Persing DH: Babesiosis. 24. Zintl A, Finnerty EJ, Murphy TM, de Waal T, Gray JS: Babesias of red deer Clin Microbiol Rev 2000, 13(3):451–469. (Cervus elaphus) in Ireland. Vet Res 2011, 42:7. 47. Schorn S, Pfister K, Reulen H, Mahling M, Silagh C: Occurrence of Babesia 25. Ferrer D, Castellà J, Gutierrez JF, Lavin S, Ignasi M: Seroprevalence of spp., Rickettsia spp. and Bartonella spp. in Ixodes ricinus in Bavarian Babesia ovis in Spanish ibex (Capra pyrenaica) in Catalonia, Northeastern public parks, Germany. Parasit Vectors 2011, 4:135. Spain. Vet Parasitol 1998, 75(2–3):93–98. 48. Semenza JC, Menne B: Climate change and infectious diseases in Europe. 26. Iori A, Gabrielli S, Calderini P, Moretti A, Pietrobelli M, Tampieri MP, Galuppi R, Lancet Infect Dis 2009, 9:365–375. Cancrini G: Tick reservoirs for Piroplasms in central and northern Italy. 49. Gortazar C, Ferroglio E, Hofle U, Frolich K, Vicente J: Diseases shared Vet Parasitol 2010, 170:291–296. between wildlife and livestock: a European perspective. Eur J Wildl Res 27. Laird JS, Kocan AA, Kocan KM, Presley SM, Hair JA: Susceptibility of 2007, 53:241–256. Amblyomma americanum to natural and experimental infections with 50. Daszak P, Cunningham AA, Hyatt AD: Emerging infectious diseases of Theileria cervi. J Wildl Dis 1988, 24(4):679–683. wildlife: threats to biodiversity and human health. Science 2000, 28. Chae JS, Waghela SD, Craig TM, Kocan AA, Wagner GG, Holman PJ: Two 287(5452):443–449. Theileria cervi SSU RRNA gene sequence types found in isolates from 51. Cassini R, Bonoli C, Montarsi F, Tessarin C, Marcer F, Galuppi R: Detection of white-tailed deer and elk in North America. J Wildl Dis 1999, Babesia EU1 in Ixodes ricinus ticks in Northern Italy. Vet Parasitol 2010, 35(3):458–465. 171(1–2):151–154. 29. Bastian S, Jouglin M, Brisseau N, Malandrin L, Klegou G, L’Hostis M, Chauvin A: 52. Randi E: Management of wild ungulate populations in Italy: captive- Antibody prevalence and molecular identification of Babesia spp. in Roe breeding, hybridisation and genetic consequences of translocations. deer in France. J Wildl Dis 2012, 48(2):416–424. Vet Res Comm 2005, 29(Suppl 2):71–75. 30. Hoby S, Robert N, Mathis A, Schmid N, Meli ML, Hofmann-Lehmann R, 53. Cantu AC, Ortega AS, Garcia-Vasquez Z, Mosqueda J, Henke SE, George JE: Lutz H, Deplazes P, Ryser-Degiorgis MP: Babesiosis in free-living chamois Epizootiology of Babesia bovis and Babesia bigemina in free-ranging (Rupicapra rupicapra) from Switzerland. Vet Par 2007, 148:341–345. white-tailed deer in northeastern Mexico. J Parasitol 2009, 95:536–542. 31. Hoby S, Mathis A, Doherr MG, Robert N, Ryser-Degiorgis MP: Babesia 54. Mattioli S, Meneguz PG, Brugnoli A, Nicoloso S: Red deer in Italy: recent – capreoli infections in Alpine chamois (Rupicapra rupicapra), roe deer changes in range and numbers. Hystrix It J Mamm 2001, 2(1):27 35. (Capreolus capreolus) and red deer (Cervus elaphus) from Switzerland. 55. Bergagna S, Zoppi S, Ferroglio E, Gobetto M, Dondo A, Di Giannatale E, J Wildlife Dis 2009, 45(3):748–753. Gennero MS, Grattarola C: Epidemiologic Survey for Brucella suis Biovar 2 32. Focardi S, Pelliccioni E, Petrucco R, Toso S: Spatial patterns and density in a Wild Boar (Sus scrofa) Population in Northwest Italy. J Wildl Dis 2009, – dependence in the dynamics of a roe deer (Capreolus capreolus) 45(4):1178 1181. population in central Italy. Oecologia 2002, 130(3):411–419. 56. Pandini W, Cesaris C: Homerange and habitat use of roe deer 33. Zintl A, Mulcahy G, Skerrett HE, Taylor SM, Gray JS: Babesia divergens,a (Capreolus capreolus) reared in captivity and released in the wild. – – bovine blood parasite of veterinary and zoonotic importance. Hystrix It J Mamm 1997, 9(1 2):45 50. Clin Microbiol Rev 2003, 16:622–636. 57. de León AA P, Strickman DA, Knowles DP, Fish D, Thacker E, de la Fuente J, 34. Cardoso L, Cortes HCE, Reis A, Rodrigues P, Simões M, Lopes AP, Vila-Viçosa MJ, Krause PJ, Wikel SK, Miller RS, Wagner GG, Almazán C, Hillman R, Messenger MT, Talmi-Frank D, Eyal O, Solano-Gallego L, Baneth G: Prevalence of Babesia Ugstad PO, Duhaime RA, Teel PD, Ortega-Santos A, Hewitt DG, Bowers EJ, microti-like infection in red foxes (Vulpes vulpes) from Portugal. Vet Parasitol Bent SJ, Cochran MH, McElwain TF, Scoles GA, Suarez CE, Davey R, Howell 2013, 196(1–2):90–95. Freeman JM, Lohmeyer K, Li AY, Guerrero FD, Kammlah DM, Phillips P, 35. The IUCN Red List of Threatened Species. http://www.iucnredlist.org/. Pound JM, Group for Emerging Babesioses and One Health Research and Development in the U.S: One Health approach to identify research needs in 36. Criado-Fornelio A, Rey-Valeiron C, Buling A, Barba-Carretero JC, Jefferies R, bovine and human babesioses: workshop report. Parasit Vectors 2010, 3(1):36. Irwin P: New advances in molecular epizootiology of canine hematic protozoa from Venezuela, Thailand and Spain. Vet Parasitol 2007, 144:261–269. doi:10.1186/1756-3305-7-70 37. Gimenez C, Casado N, Criado-Fornelio A, de Miguel FA, Dominguez-Penafiel G: Cite this article as: Zanet et al.: Piroplasmosis in wildlife: Babesia and Theileria affecting free-ranging ungulates and carnivores in the Italian A molecular survey of Piroplasmida and Hepatozoon isolated from domestic Alps. Parasites & Vectors 2014 7:70. and wild animals in Burgos (Northern Spain). Vet Parasitol 2009, 26:147–150. 38. Dezdek D, Vojta L, Curkovic S, Lipej Z, Mihaljevic Z, Cvetnic Z, Beck R: Molecular detection of Theileria annae and Hepatozoon canis in foxes (Vulpes vulpes) in Croatia. Vet Parasitol 2010, 172:333–336. 39. Penzhorn BL: Babesiosis of wild carnivores and ungulates. Vet Parasitol 2006, 138:11–21. 40. Hunfeld KP, Hildebrandt A, Gray JS: Babesiosis: Recent insights into an ancient disease. Int J Parasitol 2008, 38:1219–1237. 41. Gubbels JM, de Vos AP, van der Weide M, Viseras J, Schouls LM, de Vries E, Jongejan F: Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridization. J Clin Microbiol 1999, 37(6):1782–1789. 42. Centeno-Lima S, Do Rosário V, Parreira R, Maia AJ, Freudenthal AM, Nijhof Submit your next manuscript to BioMed Central AM, Jongejan F: A fatal case of human babesiosis in Portugal: molecular and take full advantage of: and phylogenetic analysis. Trop Med Int Health 2003, 8(8):760–764. 43. Schnittger L, Yin H, Qi B, Gubbels MJ, Beyer D, Niemann S, Jongejan F, • Convenient online submission Ahmed JS: Simultaneous detection and differentiation of Theileria and Babesia parasites infecting small ruminants by reverse line blotting. • Thorough peer review Parasitol Res 2004, 92(3):189–196. • No space constraints or color ﬁgure charges 44. García-Sanmartín J, Aurtenetxe O, Barral M, Marco I, Lavin S, García-Pérez AL, • Immediate publication on acceptance Hurtado A: Molecular detection and characterization of piroplasms infecting cervids and chamois in Northern Spain. Parasitol 2007, • Inclusion in PubMed, CAS, Scopus and Google Scholar – 134:391 398. • Research which is freely available for redistribution 45. Nagore D, García-Sanmartín J, García-Pérez AL, Juste RA, Hurtado A: Identification, genetic diversity and prevalence of Theileria and Babesia Submit your manuscript at www.biomedcentral.com/submit